OsJRL40, a Jacalin-Related Lectin Gene, Promotes Salt Stress Tolerance in Rice

High salinity is a major stress factor affecting the quality and productivity of rice (Oryza sativa L.). Although numerous salt tolerance-related genes have been identified in rice, their molecular mechanisms remain unknown. Here, we report that OsJRL40, a jacalin-related lectin gene, confers remarkable salt tolerance in rice. The loss of function of OsJRL40 increased sensitivity to salt stress in rice, whereas its overexpression enhanced salt tolerance at the seedling stage and during reproductive growth. β-glucuronidase (GUS) reporter assays indicated that OsJRL40 is expressed to higher levels in roots and internodes than in other tissues, and subcellular localization analysis revealed that the OsJRL40 protein localizes to the cytoplasm. Further molecular analyses showed that OsJRL40 enhances antioxidant enzyme activities and regulates Na+-K+ homeostasis under salt stress. RNA-seq analysis revealed that OsJRL40 regulates salt tolerance in rice by controlling the expression of genes encoding Na+/K+ transporters, salt-responsive transcription factors, and other salt response-related proteins. Overall, this study provides a scientific basis for an in-depth investigation of the salt tolerance mechanism in rice and could guide the breeding of salt-tolerant rice cultivars.


Introduction
Rice (Oryza sativa L.) is the main agricultural crop in the world and the staple food of most of the global population [1]. Salt stress is one of the important factors affecting rice production. Approximately 7% of the total land area is affected by high salinity [2,3]. Excess salt adversely affects many plant processes including photosynthesis, metabolism, and respiration, resulting in stunted growth and even death [4]. High salt can also cause Na + poisoning, which seriously affects plant growth and development and consequently reduces grain quality and yield in rice. Plants respond to the unfavorable environment surrounding them by generating complex inter-gene interactions and cross-talk with different molecular pathways [5]. How plants adapt to salt stress and how their salt tolerance levels can be improved further have long been the hot topics of research in the field of plant physiology and ecology.
Plants have evolved several mechanisms of salt tolerance, including membrane modification, ion absorption, transport, and separation, and salt excretion, etc. [6]. Many genes and gene families have been reported to play important roles in plant salt tolerance. For example, OsZFP213 and OsMADS25 are involved in regulating the maintenance of the antioxidant system, which improves the salt tolerance of rice by reducing the electrical conductivity and the accumulation of harmful substances such as hydrogen peroxide and malondialdehyde (MDA) [7,8]. High-affinity K + transporter (KHT) [9,10], Na + /H + antiporter (NHX) [11,12], and high-affinity K + (HAK) transporter [13] gene families regulate Na + -K + balance under salt stress, thus promoting salt tolerance. In addition, plant  OsJRL40 and JRLs in other species (rice, Arabidopsis, wheat, maize, and soybean). The phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.
Next, we conducted the phylogenetic analysis of JRL proteins of rice, maize, wheat, Arabidopsis, and soybean, whose protein sequences are derived from Song et al. [19]. OsJRL40 was found to be most closely related to maize ( Figure 1B).

Expression and Subcellular Localization Assay of OsJRL40
To verify the function of OsJRL40, real-time fluorescence quantitative PCR (RT-qPCR) analysis was performed. The expression of OsJRL40 was most strongly induced when treated with 100 mM NaCl, reaching a peak at 6 h and then declining, indicating that OsJRL40 is a salt stress-responsive gene (Figure 2A). OsJRL40 was also induced in response to exogenous abscisic acid (ABA) application ( Figure 2B). Next, we analyzed the OsJRL40 expression pattern in different tissues of japonica rice Nip by RT-qPCR. OsJRL40 showed a higher expression level in the stem than in other tissues ( Figure 2C). To further examine the expression pattern of OsJRL40 in different tissues of rice plants, a 2.5-kb OsJRL40 promoter fragment driving the β-glucuronidase (GUS) reporter gene was introduced into the rice. GUS staining was detected in roots, stems, leaves, internodes, bud sheaths, and panicles. The cross-sections of roots and leaves revealed staining of the xylem and vascular systems, respectively ( Figure 2D).  To investigate the subcellular localization of OsJRL40, the OsJRL40-GFP fusion was transiently expressed in Nip protoplasts under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter. Green fluorescence was observed in the cytoplasm ( Figure 2E), indicating that OsJRL40 is a cytosolic protein.

OsJRL40 Positively Regulates Salt Stress Tolerance in Rice at the Seedling Stage
In a previous study, we preliminarily determined that OsJRL40 is involved in regulating salt tolerance in rice [38]. To investigate the possible function of OsJRL40 in the salt stress response, two OsJRL40 loss-of-function knockout mutant lines (KO1 and KO2) were generated in the Nip background using the CRISPR/Cas9 technology. The KO1 mutant contained a 1-nt deletion in the first exon of OsJRL40, causing premature termination of protein, while the KO2 mutant contained a 1-nt insertion in the second exon of OsJRL40, resulting in frameshift mutation ( Figure 3A). We also generated two independent OsJRL40 overexpression lines (OE1 and OE2) in the Nip background. RT-qPCR indicated that OsJRL40 was significantly up-regulated in both OE1 and OE2 compared with the WT (Nip) ( Figure 3B). Next, we evaluated the salt tolerance of WT, KO, and OE plants at the seedling stage. The KO1 and KO2 seedlings showed severe wilting, while the OE1 and OE2 seedlings grew better than the WT ( Figure 3C). Compared with the WT, the survival rate, fresh weight, and chlorophyll content of KO1 and KO2 lines were significantly lower, while those of OE1 and OE2 were higher after the NaCl treatment ( Figure 3D-F). These results indicate that OsJRL40 positively regulates salt stress tolerance in rice.

OsJRL40 Enhances Salt Tolerance in Rice at the Reproductive Stage
To determine whether OsJRL40 affects salt tolerance in rice at the reproductive stage, we examined the phenotypes and agronomic traits of WT, KO, and OE plants under normal (0 mM NaCl) and salt stress (150 mM NaCl) conditions ( Figure 4). Under normal conditions, the thousand-grain weight of KO plants was lower, while that of OE plants was higher than the WT ( Figure 4H), and no significant differences were observed in the other traits ( Figure 4). Under salt stress conditions, all KO plants died, while the OE lines showed significantly greater plant height, panicle length, seed setting rate, and thousand-grain weight than the WT ( Figure 4D,F-H). The WT, KO, and OE plants showed no significant difference in panicle number under salt stress conditions ( Figure 4E). These results suggest that OsJRL40 enhances the salt tolerance and yield of reproductive-stage rice plants under salt stress.

ABA Affects Seed Germination and Plant Growth of OE Lines
According to previous research [43], overexpression of JRL family genes increases the sensitivity of plants to ABA and alters the expression of downstream genes. RT-qPCR analysis revealed that the OsJRL40 was responsive to exogenous ABA ( Figure 2B). Under normal conditions, the germination rate of KO and OE seeds was similar to that of WT seeds. However, in the 2 µM ABA treatment, the germination rate of KO seeds was higher than that of WT seeds, whereas the germination rate of OE seeds was lower than that of WT seeds. In the 5 µM ABA treatment, the seed germination rate of KO lines was approximately 30%, whereas that of OE seeds was almost 0 ( Figure 5A,B).

ABA Affects Seed Germination and Plant Growth of OE Lines
According to previous research [43], overexpression of JRL family genes increases t sensitivity of plants to ABA and alters the expression of downstream genes. RT-qPC analysis revealed that the OsJRL40 was responsive to exogenous ABA ( Figure 2B). Und normal conditions, the germination rate of KO and OE seeds was similar to that of W seeds. However, in the 2 μM ABA treatment, the germination rate of KO seeds was high than that of WT seeds, whereas the germination rate of OE seeds was lower than that WT seeds. In the 5 μM ABA treatment, the seed germination rate of KO lines was appro imately 30%, whereas that of OE seeds was almost 0 ( Figure 5A, B).
To further investigate the relationship between OsJRL40 expression and exogeno ABA application, we germinated the WT, OE, and KO seeds on half-strength Murash and Skoog (1/2 MS) medium for 2 days and then transferred them to 1/2 MS mediu containing varying concentrations of ABA to evaluate seedling growth. On normal m dium, the growth of the KO and OE lines was similar to that of WT plants ( Figure 5C- To further investigate the relationship between OsJRL40 expression and exogenous ABA application, we germinated the WT, OE, and KO seeds on half-strength Murashige and Skoog (1/2 MS) medium for 2 days and then transferred them to 1/2 MS medium containing varying concentrations of ABA to evaluate seedling growth. On normal medium, the growth of the KO and OE lines was similar to that of WT plants ( Figure 5C-E). However, in the presence of 2 and 5 µM ABA, the roots and shoots of WT plants were shorter than those of KO lines and longer than those of OE lines ( Figure 5C-E). These results indicate that overexpression of OsJRL40 increases the ABA sensitivity of rice plants, whereas knockout mutation of OsJRL40 has the opposite effect.

OsJRL40 Enhances the Antioxidant Capacity of Rice under Salt Stress
To explore the molecular mechanism of OsJRL40-induced salt stress tolerance in r we studied the physiological and biochemical parameters of WT, KO, and OE plants der salt stress. No significant differences were observed among the three genotypes un normal conditions. However, a 2 d treatment with 100 mM NaCl led to the accumula of reactive oxygen species (ROS) as revealed by diaminobenzidine (DAB) staining, wh was stronger in KO mutants than in the WT ( Figure 6A, B). Additionally, the conten

OsJRL40 Enhances the Antioxidant Capacity of Rice under Salt Stress
To explore the molecular mechanism of OsJRL40-induced salt stress tolerance in rice, we studied the physiological and biochemical parameters of WT, KO, and OE plants under salt stress. No significant differences were observed among the three genotypes under normal conditions. However, a 2 d treatment with 100 mM NaCl led to the accumulation of reactive oxygen species (ROS) as revealed by diaminobenzidine (DAB) staining, which was stronger in KO mutants than in the WT ( Figure 6A,B). Additionally, the content of hydrogen peroxide (H 2 O 2 ) was higher in KO mutant leaves than in WT leaves ( Figure 6D). The malondialdehyde (MDA) content and relative electrical conductivity (EC) were significantly higher in KO mutants ( Figure 6C,E), which is consistent with their performance under salt stress. The trends displayed by OE plants, in terms of ROS and MDA contents and EC, were opposite to those displayed by KO plants. The activities of ROS scavenging enzymes are closely related to the salt stress tolerance levels of plants [44]. Consistently, the activities of superoxide dismutase (SOD) and peroxidase (POD) were dramatically lower in KO1 and KO2 lines and significantly higher in OE1 and OE2 lines than in the WT ( Figure 6F,G). These results indicate that OsJRL40 improves antioxidant enzyme activity in rice under salt stress.

OsJRL40 Regulates Na + -K + Homeostasis under Salt Stress
To test whether the growth phenotype of KO1 and KO2 lines under high salinity conditions was caused by the overaccumulation of Na + , we determined the Na + and K + contents of the roots and shoots of WT, KO1, KO2, OE1, and OE2 plants. No differences in Na + and K + contents were detected among the different genotypes under normal conditions. However, under salt stress conditions, the KO1 and KO2 plants accumulated more Na + ( Figure 7A,D), but less K + ( Figure 7B,E), in shoots and roots than the WT plants, while the OE plants showed the opposite results. The Na + /K + ratio was significantly higher in the shoots and roots of KO1 and KO2 plants than in those of WT, OE1, and OE2 plants ( Figure 7C,F). Taken together, these results suggest that OsJRL40 enhances salt tolerance in rice by reducing Na + accumulation in the shoot and root, thus modifying the Na + /K + ratio.

OsJRL40 Mediates Salt Tolerance in Yeast
To further clarify the function of OsJRL40, we performed functional assays in yeast (Saccharomyces cerevisiae) strain BY4741. The plasmid containing OsJRL40 was transformed into BY4741 cells, and the transformed cells were grown on media containing different concentrations of NaCl ( Figure 8). Under normal conditions (0% NaCl), no significant difference was detected between yeast transformants carrying the empty pYES2 vector (control) and those expressing OsJRL40 (Figure 8). Under salt stress conditions, the growth of BY4741 cells carrying the empty vector was inhibited more significantly than that of yeast cells expressing OsJRL40, and the growth inhibition of yeast cells increased with the increase in NaCl concentration ( Figure 8). These results suggest that OsJRL40 enhances the salt tolerance of yeast.

OsJRL40 Affects the Expression Profiles of Stress-Responsive Genes
To further clarify the mechanism of action of OsJRL40 at the molecular level, we performed RNA-seq analysis on WT and KO1 plants grown under normal (0 mM NaCl) or salt stress (100 mM NaCl) conditions. A total of 2533 genes showing differential expression between WT and KO1 plants were identified, of which 446 were up-regulated and 1406 were down-regulated in KO1 plants compared with the WT (Figure 9A). Under normal conditions, 254 genes were up-regulated and 229 genes were down-regulated in KO1 plants compared with the WT (Figure 10A). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these differentially expressed genes (DEGs) were involved in plant-pathogen interaction, sesquiterpenoid and triterpenoid biosynthesis, and mitogenactivated protein kinase (MAPK) signaling pathway ( Figure 9B), and Gene Ontology (GO) enrichment analysis showed that these DEGs were involved in defense response, DNA integration, and ADP binding ( Figure 9C). Under salt stress conditions, 192 genes were up-regulated and 1177 genes were down-regulated in KO1 plants compared with the WT ( Figure 10B). KEGG enrichment analysis showed that these DEGs were enriched in plant-pathogen interaction, flavonoid biosynthesis, phenylpropanoid biosynthesis, and MAPK signaling pathway ( Figure 10C). GO enrichment analysis showed that most of these DEGs were involved in response to wounding, defense response, stress regulation, and so on ( Figure 10D). enzymes are closely related to the salt stress tolerance levels of plants [44]. Consistent the activities of superoxide dismutase (SOD) and peroxidase (POD) were dramatica lower in KO1 and KO2 lines and significantly higher in OE1 and OE2 lines than in the W ( Figure 6F, G). These results indicate that OsJRL40 improves antioxidant enzyme activ in rice under salt stress.  the OE plants showed the opposite results. The Na + /K + ratio was significantly higher the shoots and roots of KO1 and KO2 plants than in those of WT, OE1, and OE2 pla ( Figure 7C, F). Taken together, these results suggest that OsJRL40 enhances salt toleran in rice by reducing Na + accumulation in the shoot and root, thus modifying the Na + ratio. Figure 7. Effects of OsJRL40 on Na + and K + accumulation in rice seedlings under salt stress con tions. Fifteen-day-old seedlings were treated with a hydroponic medium containing 100 mM N for 2 days. (A, B) Contents of Na + (A) and K + (B) in shoots. (C) Na + /K + ratio in shoots. (D, E) Conte of Na + (D) and K + (E) in roots. (F) Na + /K + ratio in roots. Data represent mean ± SD (n = 3). Asteri indicate statistically significant differences (Student's t-test; * p < 0.05, ** p < 0.01). DW, dry weig

OsJRL40 Mediates Salt Tolerance in Yeast
To further clarify the function of OsJRL40, we performed functional assays in ye (Saccharomyces cerevisiae) strain BY4741. The plasmid containing OsJRL40 was transform into BY4741 cells, and the transformed cells were grown on media containing differe concentrations of NaCl ( Figure 8). Under normal conditions (0% NaCl), no significant d ference was detected between yeast transformants carrying the empty pYES2 vector (co trol) and those expressing OsJRL40 (Figure 8). Under salt stress conditions, the growth BY4741 cells carrying the empty vector was inhibited more significantly than that of ye cells expressing OsJRL40, and the growth inhibition of yeast cells increased with the crease in NaCl concentration (Figure 8). These results suggest that OsJRL40 enhances t salt tolerance of yeast.  . Effects of OsJRL40 on Na + and K + accumulation in rice seedlings under salt stress conditions. Fifteen-day-old seedlings were treated with a hydroponic medium containing 100 mM NaCl for 2 days. (A,B) Contents of Na + (A) and K + (B) in shoots. (C) Na + /K + ratio in shoots. (D,E) Contents of Na + (D) and K + (E) in roots. (F) Na + /K + ratio in roots. Data represent mean ± SD (n = 3). Asterisks indicate statistically significant differences (Student's t-test; * p < 0.05, ** p < 0.01). DW, dry weight.
Because most of the DEGs were down-regulated after salt treatment and most of them were involved in stress response, we focused on these down-regulated genes. A comparison of the DEGs identified in KO plants before and after salt stress treatment revealed that 1021 DEGs were only down-regulated in KO plants. These down-regulated genes were defined as the candidate targets of OsJRL40 ( Figure 11A) and were further annotated by KEGG and GO enrichment analyses. The results of KEGG enrichment analysis showed that most of these DEGs were involved in plant-pathogen interaction, hormone signal transduction, MAPK signaling pathway, and the biosynthesis and metabolism of phenylpropanoids, starch and sucrose, and flavonoids. The results of GO enrichment analysis showed that most of the genes were involved in stress response, stress regulation, and bind activity, and some genes were involved in the salt stress response regulation pathway ( Figure 11B,C). We focused on some genes involved in stress response pathways, including transcription factor genes (WARKY54, WARKY71, MYB2, and NAC19), Na + -K + ion transporter genes (OsHAK1, OsHAK17, OsKAT1, OsAKT1, and OsAKT2), and some salt stress response-related genes. Most of these genes have been reported to respond to salt stress and play an important role in salt tolerance. The OsHAK1 regulates salt tolerance in rice by regulating K + concentration [13]. The OsKAT1 confers salinity tolerance to yeast and rice cells [45]. The expressing NAC19 gene increased the salt tolerance of plants, and the leaf water content and chlorophyll content of these plants were higher than those of the WT [46]. OsWRKY54 regulates the expression of some essential genes related to salt tolerance [47]. The expression of the OsWRKY71 transcript level was the highest under salinity stress [48]. OsGAP1 attenuates salt stress and binds to phospholipids and the unconventional G protein OsYCHF1 [49]. trol) and those expressing OsJRL40 (Figure 8). Under salt stress conditions, the growth BY4741 cells carrying the empty vector was inhibited more significantly than that of ye cells expressing OsJRL40, and the growth inhibition of yeast cells increased with the crease in NaCl concentration (Figure 8). These results suggest that OsJRL40 enhances salt tolerance of yeast.   To confirm the results of RNA-seq analysis, we selected eight genes for RT-qPCR analysis. Our RT-qPCR results indicated that these genes were up-regulated in WT plants and down-regulated in KO plants under salt stress ( Figure 12A), which was consistent with our RNA-seq results. Collectively, RNA-seq analysis revealed that OsJRL40 regulates salt tolerance in rice by regulating Na + -K + transport-related genes, salt response-associated transcription factor genes, and other salt response-related genes.  WT, KO1, KO2, OE1, and OE2 seedlings at the three-leaf stage were treated with 100 mM NaCl for 8 h. After RNA extraction and reverse transcription, RT-qPCR was performed. Osactin was used as the internal control gene. Data represent the mean ± SD of three independent replicates. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01).

Discussion
The JRL family is the third largest family of lectins in rice. Transcriptome and whole- After RNA extraction and reverse transcription, RT-qPCR was performed. Osactin was used as the internal control gene. Data represent the mean ± SD of three independent replicates. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01).

Discussion
The JRL family is the third largest family of lectins in rice. Transcriptome and wholegenome sequencing analyses show that JRL family genes are involved in the salt stress response, although the experimental evidence available is limited. Here, we provide genetic evidence demonstrating that the JRL family gene OsJRL40 positively regulates tolerance to salt stress in rice. OsJRL40 was mainly expressed in nodes and responded quickly to salt stress. The osjrl40 KO mutants were sensitive to salt stress, while OsJRL40 OE lines exhibited enhanced salt stress tolerance. In addition, the OE plants showed strong salt tolerance at the reproductive stage, as evident from their increased panicle length, seed setting rate, thousand-grain weight, and consequent yield under salt stress conditions. Under salt stress conditions, OsJRL40 overexpression enhanced the antioxidant capacity of cells and eliminated excessive ROS accumulation. In addition, OsJRL40 regulated the Na + -K + balance and the expression of many stress-related genes, suggesting that OsJRL40 plays an important role in salt stress tolerance.
In most plant species, salt stress tolerance is associated with a decrease in Na + accumulation in aerial plant parts. Plants have evolved various mechanisms to enhance salt tolerance, including increasing the Na + -chelating capacity of vacuoles and promoting the transfer of Na + from bud to root via the phloem sap [50]. According to previous studies, K + channel proteins including HAKs, AKTs, and KATs play important roles in salt stress tolerance in rice [51,52]. HAK family genes regulate K + uptake and translocation, salt tolerance, and osmotic potential regulation [13,53]. The expression of OsAKTs and OsKATs is induced in the root and stem tissues of rice in response to salt stress. The K + transporter OsHAK1 regulates salt tolerance in rice by regulating K + concentration [48]. AKT1, AKT2, and KAT1 are induced by salt stress [54]. The rice Shaker K + channel protein OsKAT1 confers salinity tolerance to yeast and rice cells [45]. While the loss-of-function mutation of OsJRL40 increased the Na + content of shoots and roots, its overexpression had the opposite effect. This suggests that OsJRL40 regulates K + transporters to maintain Na + -K + homeostasis in rice under salt stress conditions. RT-qPCR results showed that, under salt stress conditions, the expression levels of OsHAK1, OsAKT1, and OsKAT1 were significantly lower in KO mutant plants and significantly higher in OE plants than in the WT. These results will serve as a reference for in-depth research on the salt tolerance mechanism of JRL family genes.
RNA-seq analysis showed that 1406 and 162 genes were down-regulated in the KO mutant and OE plants, respectively, compared with the WT. KEGG enrichment analysis demonstrated that most DEGs were involved in plant hormone signaling and MAPK signaling pathways and substance anabolic pathways. GO functional analysis showed that most DEGs were involved in stress response regulation. Our results showed that OsJRL40 was also responsive to exogenous ABA application, and the OE lines were sensitive to ABA. Previous research shows that members of the OsJRL family regulate ABA response and the expression of genes involved in the ABA signaling pathway [43]. Poplar JRL genes mediate the response to ABA under salt stress, maintaining ROS and ion homeostasis [37]. Thus, it is possible that the rice JRL family genes impart salt tolerance by participating in the ABA signaling pathway. Our RT-qPCR showed that the expression of some salt tolerance genes, including OsWARKY71, OsMYB2, OsNAC19, OsJAC1, and OsCDPK13, was enhanced by OsJRL40. This suggests that the participation of OsJRL40 in salt tolerance may be related to the regulation of the expression of these genes. OsNAC2, a member of the NAC transcription factor family, was strongly induced by ABA and osmotic stresses such as drought and high salt [55]. The expressing NAC19 gene increased drought and salt tolerance of plants, and the leaf water content and chlorophyll content of these plants were higher than those of the WT [46]. OsWRKY54 regulates the expression of some essential genes related to salt tolerance [47]. The expression of the OsWRKY71 gene was up-regulated in response to low temperature, salinity, drought, and injury, and its transcript level was the highest under salinity stress [48]. Calmodulin (CAM) subtypes mediate salt-induced Ca 2+ signaling through the activation of MYB transcriptional activators, leading to salt tolerance in plants [56]. OsGAP1 attenuates salt stress, stimulates a defense response, and binds to phospholipids and the unconventional G protein OsYCHF1 [49]. These results suggest that the involvement of OsJRL40 in salt tolerance is attributable to the regulation of the expression of these genes.
Overall, this study shows that the JRL family gene OsJRL40 induces salt tolerance in rice by regulating some critical Na + /K + transporters, upstream regulatory factors, and stress-related genes.

Plant Material and ABA Treatment
Rice (Oryza sativa ssp. japonica) cultivar Nipponbare (Nip) was used as the WT in this study. After germination, the seeds were transferred to a normal hydroponic medium, and seedlings were grown to the three-leaf stage over a period of approximately 15 d. Then, the seedlings were grown in 100 mM NaCl for 4 d and subsequently returned to the normal hydroponic medium to recover for 7 d. Finally, the survival rate, fresh weight, and chlorophyll content of seedlings were determined. To observe the salt-tolerant phenotype of rice plants at the reproductive stage, plants at the tillering stage were transferred to 150 mM NaCl-containing soil and grown to maturity before quantifying the agronomic traits. To understand the effect of ABA treatment on rice, seeds and seedlings of WT and mutants (KO1, KO2, OE1, and OE2), they were treated with 0, 2, and 5 µM ABA, respectively. Referring to the previous research and modifying it slightly [57], seeds were inoculated on 1/2 MS medium supplemented with different concentrations of ABA (0, 2, 5 mM) for 5 days. To observe seedling phenotypes, budding seedlings were transferred to 1/2 MS medium supplemented with different concentrations of ABA (0, 2, 5 mM) for about 7 days. Then the phenotype was observed and the root length and shoot length were measured.

Generation of Mutant and Transgenic Lines
The CRISPR/Cas9 method was used to create the osjrl40 KO mutants. According to the criteria described by Maio and colleagues [58], two 20-bp target sites (1: GGACGT-GATCGCCATGGTGG; 2: GGAAAGGGTTGCATCGTCGT) were selected. The CRISPR-Cas9-OsJRL40 constructs were then transformed into Nip plants via Agrobacterium-mediated transformation. The resultant mutant lines were screened for hygromycin resistance and then confirmed by PCR using sequence-specific primers. Two KO mutant lines, KO1 and KO2, were obtained. Primers used for the generation and identification of KO lines are listed in Table S1.
To construct the OsJRL40 overexpression plants (OE1 and OE2), the open reading frame of OsJRL40 was amplified by PCR from rice cDNA using the sequence-specific primer pair 1390-OsJRL40-F/R (Table S1). The purified PCR product was cloned into the pCAMBIA1390-Ubi vector using SpeI and BamHI restriction endonucleases. To generate plants expressing the pOsJRL40: GUS construct, the OsJRL40 promoter (2500 bp upstream of ATG) was amplified by PCR from Nip genomic DNA using the primer pair 1301-OsJRL40-F/R (Table S1). The PCR product was cloned into the pCAMBIA1301 binary vector. The resultant plasmid was introduced into Agrobacterium tumefaciens strain EHA105, which was then used to transform Nip plants as described previously [59].

Gene Expression Analysis
Total RNA was extracted from whole plants using the TRIzol Reagent (Invitrogen, Waltham, MA, USA) and subjected to reverse transcription to synthesize cDNA. Then, RT-qPCR was performed on the ABI PRISM 7500 RT-qPCR instrument (Applied Biosystems, Waltham, MA, USA) using the Takara quantitative kit RR420A and sequence-specific primers, which were designed with Primer Premier 5 and synthesized by Sangon Biotech (Shanghai, China). The Osactin gene was used as an internal reference.

GUS Staining Assay
GUS staining was performed as described previously [60]. Briefly, the tissues of ProOsJRL40-GUS transgenic rice were incubated overnight in a GUS staining solution at 37 • C. Then, chlorophyll was removed using 75% ethanol. Finally, the tissues were examined and photographed using the Olympus SZX7 microscope.

DAB Staining Assay
As described previously [61], Leaves were collected from rice plants before and after the NaCl stress treatment. The leaf samples were incubated in 1 mg mL −1 3,3 -DAB solution (containing 0.05% Tween and 10 mM Na 2 HPO 4 [pH 3.8]) at 37 • C overnight. The stained leaves were discolored with ethanol. Finally, the leaves are transferred to fresh ethanol and imaged.

Electrolyte Leakage Assay
The relative conductivity is as described previously [38]. Rice leaves are washed with deionized water, and the surface water was soaked dry. Next, 25 mL of deionized water was added to the test tube containing the sample and incubated at room temperature for 1 h. The EC of the leachate was measured with a conductivity meter as the initial conductivity (al), and that of the extract (a2) was measured at 0, 2, 4, 6, 8, 10, 12, and 24 h. Finally, the samples were boiled for 15 min. The value of absolute EC (EC after boiling; a3). To determine the cell membrane permeability, relative EC (%) was calculated using the equation below:

Measurement of Antioxidant Enzyme Activities and H 2 O 2 Content
Plants of all genotypes (WT, KO1, KO2, OE1, and OE2) were grown in normal hydroponic solution for 14 d and then treated with 100 mM NaCl for 2 d. The activities of SOD and POD and the content of H 2 O 2 were measured using the corresponding assay kits (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's instructions.

Measurement of MDA and Chlorophyll Contents
To measure the MDA content, an indicator of the degree of lipid peroxidation [62], fresh rice leaves were ground in trichloroacetic acid on ice. After centrifugation, the supernatant was added to thiobarbituric acid, and the sample was boiled in a water bath for 15 min. After cooling to room temperature, the absorbance of the sample was measured at 450, 532, and 600 nm. To determine the chlorophyll content of leaves, chlorophyll was extracted from leaves (100 mg), and the absorbance of the extract was measured at 663 and 645 nm [38,61].

Subcellular Localization Analysis
To analyze the subcellular localization of OsJRL40, the CDS of OsJRL40 minus the stop codon was amplified from Nip genomic DNA using gene-specific primers (Table  S1), digested with PstI, and cloned into the PstI-digested pCAMBIA1390-GFP vector. The resultant 35S:OsJRL40-GFP construct was transiently expressed in rice protoplasts via polyethylene glycol (PEG)-mediated transformation [63]. The GFP signal was detected using the Zeiss LSM 880 microscope [64].

Yeast Complementation Assay
The OsJRL40 CDS was amplified using gene-specific primers (Table S1), and then cloned into the pYES2 vector using HindIII and XbaI restriction enzymes. The resultant vector was introduced into BY4741 yeast cells via the LiAC/ssDNA/PEG-mediated transformation method. Yeast growth was observed on a uracil-deficient medium containing 2% galactose (carbon source) and 2%, 5%, or 8% NaCl.

Ion Content Analysis
Whole plants of all genotypes (WT, KO1, KO2, OE1, and OE2) were ground with a plant grinder, and the powdered samples were passed through a 1 mm sieve. Then, 2 mL of nitric acid-perchloric acid (5:1) mixture was added to 50 mg of the ground sample. The digested sample was filtered, and K + and Na + concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7850).

RNA-Seq Analysis
Whole WT and KO1 plants were sampled before and after the 6 h 100 mM NaCl treatment and sent to Biomarker Technologies for RNA-seq analysis. The RNA-seq data were analyzed as described by Zhang et al. [65]. Then, the DEGs were functionally annotated by performing the GO and KEGG enrichment analyses [66,67].

Statistical Analysis
Figures were constructed using GraphPad Prism5. Data were represented as the mean ± SD of three independent replicates. The mean of three replicates per treatment was calculated using the PASW statistical Software18, and statistically significant differences were detected using one-way analysis of variance (ANOVA) and Student's t-test. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data supporting the conclusions of this article are provided within the article and in its additional files and are available upon reasonable request to the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.